Project Summary Abstract: Approximately 30,000 American men die each year of metastatic castration-resistant prostate cancer (CRPC) due to the inevitable progression of resistance to first-line treatment with Docetaxel. As a unique addition to the family of microtubule-stabilizing cytotoxic agents (MSAs), zampanolide covalently binds to??-tubulin, whereas docetaxel binds non-covalently. This has opened up new avenues to more-effective chemotherapeutics for CRPC. However, limited supply of natural zampanolide from the wild constitutes a serious obstacle to support further drug development activities. Four published synthetic approaches are not efficient and CANNOT serve as its alternative source for further development as a drug candidate. More importantly, we envision that zampanolide possesses poor pharmacokinetic profiles as a drug candidate due to its chemically fragile side chain and metabolically vulnerable lactone moiety. The objective of the proposed research is therefore to develop zampanolide mimics to improve their pharmacokinetic profiles, therapeutic window, and synthetic efficiency yet retain the impressive therapeutic potency and unique mechanism of action for potentially clinical treatment of CRPC. The fundamental hypotheses of this project are: 1) that bioisosteric replacements of the N-acyl hemiaminal side chain and of the lactone in core structure of zampanolide will improve their chemically and metabolically stability yet retain the impressive cytotoxic potency as well as the unique mechanism of action; and 2) remodeling the side chain and/or simplification of the core structure will enable the syntheses to be means for a reliable and renewable source of material. The following specific studies will be carried out to test these hypotheses: 1) To establish stabilized mimics of (-)-zampanolide and practical synthetic methods. A group of zampanolide mimics, with N-acyl hemiaminal and lactone moiety remodeled, will be synthesized by practical and manageable methods. The cytotoxic and anti-proliferative potency against both drug-sensitive and resistance prostate cancer cells will be assessed; 2) To identify simplified and stabilized mimics as well as manageable synthetic methods. A group of new stabilized zampanolide mimics with a simplified core will be synthesized and evaluated; 3) To define the in vitro pharmacokinetic profiles and toxicity of promising mimics; and 4) To validate the covalently-binding mechanism of action as MSAs. This proposal is expected to result in, at least, a new stabilized zampanolide mimic with retained in vitro potency against both drug-sensitive and resistant prostate cancer cells and unique mechanism of action, as well as good in vitro pharmacokinetic and safety profiles. This proposal will bring to my research group with techniques in in vitro pharmacokinetic and mechanistic studies of drug actions and allow me to bring high quality research experiences to the undergraduate and graduate students, and position me to be competitive for SC1, R15, R01, and other NIH awards in the future.